CRISPR Gene Targeting and Genome Editing Methods Offering Increased Accuracy
With the use of CRISPR, gene targeting in experiments involving genome engineering, transgenes and gene knockout has advanced to an impressive extent. While gene targeting was always possible with various other gene editing techniques, such as TALENs and the DSB-inducing of nucleases with customized DNA binding, the advent of CRISPR/Cas9 was a refreshing change brought to the world of genetic engineering. Breakthroughs obtained by scientists like Feng Zhang and George Church have showed that CRISPR/Cas9 can be used in human cell cultures and in many other organisms as well, including plants, fruit flies, nematodes and even mice and primates.
Targeting Made Easier with CRISPR/Cas9
When it comes to the use of CRISPR, gene targeting is one of the most significant advantages of the system. Not only is CRISPR/Cas9 able to target specific genes with fewer problems and errors involved, but it also has less difficulty maintaining its accuracy and consistency throughout multiple trials. There are two main components that lead to this superiority, making CRISPR/Cas9 more straightforward and easier to use: reproducible genome cleavage and programmable sgRNA cloning. Even though some aspects that were used to compare Cas9 with other techniques have stayed the same, the differences that set CRISPR/Cas9 apart have led to the possibility of targeted double-strand breaks and improved gene editing overall.
How Is Gene Targeting Obtained?
CRISPR-Cas immunity occurs naturally in the immune response of various archaea and bacteria. Its natural role is to help the organism avoid infection and natural transformation through the introduction of foreign nucleic acids. This function is used by scientists to improve CRISPR gene targeting and achieve better results with CRISPR Type 2 systems. The Cas9 protein plays a key role in this process by determining the correct location on the genome of the host organism, and then bonding with the correct base pairs in the host DNA.
Cas9: The Simplest Method for Genome Targeting
There is a lot of talk about CRISPR, gene targeting and the way in which the two are tied together. The main answer here is the Cas9 protein, which was named as the component to one of the most accurate systems ever used for genome engineering and gene targeting. The Cas9 system was first used shortly after the development of the first CRISPR/Cas methods, and it was eventually perfected by Jennifer Doudna and Emmanuelle Charpentier, who recombined the two RNAs into a single guide RNA that could more easily target and cut the specified DNA components. As of 2015, scientists were even able to obtain effective genetic edits in human tripronuclear zygotes, marking a new stage for human genetic engineering with the help of the CRISPR/Cas9 technique.
Targeting Differences: Cas9 vs Cpf1
Regarding the use of CRISPR gene targeting, an important competitor has appeared in 2015, that continues to contest the gene targeting accuracy obtained through Cas9 techniques. Cpf1 is a newly discovered nuclease that has successfully been used as a substitute for the Cas9 protein. While results were similar, Cpf1 gene editing resulted in a more “staggered” cut and the need for only a single CRISPR RNA. Moreover, Cpf1 techniques have led to surprisingly positive results when it came to more successful target-specific DNA assembly and superior multiplexed genome editing results.
CRISPR and Genome Engineering Advancements
CRISPR is being widely used for many different purposes, and it continues to raise the bar when it comes to genetic engineering accuracy. The level of genetic targeting that CRISPR/Cas methods are capable of have made them an important research tool pertaining to fields such as biomedicine, gene function research and cancer research. Moreover, with CRISPR, new avenues are open when it comes to successful in vivo genome editing, which was obtained in a number of target organisms, including Saccharomyces cerevisiae and Arabidopsis. Due to these successes and because of the continuing commercial and research backing that it gets, CRISPR gene targeting has a bright future in the years to come.